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1. A conserved uORF impacts APOBEC3G translation and is essential for translational inhibition by the HIV-1 Vif protein

Author

Libre, Camille, Seissler, Tanja, Guerrero, Santiago, Batisse, Julien, Verriez, Cédric, Stupfler, Benjamin, Gilmer, Orian, Weber, Melanie, Cimarelli, Andrea, Etienne, Lucie, Marquet, Roland, Paillart, Jean-Christophe, Architecture et Réactivité de l'ARN (ARN), Institut de biologie moléculaire et cellulaire (IBMC), Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS), Centre International de Recherche en Infectiologie - UMR (CIRI), École normale supérieure - Lyon (ENS Lyon)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Centre International de Recherche en Infectiologie (CIRI), École normale supérieure de Lyon (ENS de Lyon)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Université Jean Monnet - Saint-Étienne (UJM)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), and MATHELIN, Sandra

Subjects
[SDV] Life Sciences [q-bio], viruses, [SDV]Life Sciences [q-bio], [SDV.BBM] Life Sciences [q-bio]/Biochemistry, Molecular Biology, virus diseases, [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, biochemical phenomena, metabolism, and nutrition, [SDV.BBM.BM] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology
Abstract

The HIV-1 Vif protein is essential for viral fitness and pathogenicity. Vif decreases expression of cellular cytosine deaminases APOBEC3G (A3G), A3F, A3D and A3H, which inhibit HIV-1 replication by inducing hypermutations during reverse transcription. Vif counteracts A3G by several non-redundant mechanisms (transcription, translation and protein degradation) that concur in reducing the levels of A3G in cell and in preventing its incorporation into viral particles. How Vif affects A3G translation remains unclear. Here, we uncovered the importance of a short conserved uORF (upstream ORF) located within two critical stem-loop structures of the 5′ untranslated region (5′UTR) of A3G mRNA. Extensive mutagenesis of A3G 5′-UTR, combined with an analysis of their translational effect in transfected cells, indicated that the uORF represses A3G translation and that A3G mRNA is translated through a dual leaky-scanning and re-initiation mechanism. Interestingly, the uORF is also mandatory for the Vif-mediated repression of A3G translation. Furthermore, we showed that the redirection of A3G mRNA into stress granules was dependent not only on Vif, but also on the uORF. Overall, we discovered that A3G translation is regulated by a small uORF conserved in the human population and that Vif uses this specific motif to repress its translation.

Published
2021

2. The IRES5′UTR of the dicistrovirus cricket paralysis virus is a type III IRES containing an essential pseudoknot structure

Author

Gross, Lauriane, Vicens, Quentin, Einhorn, Evelyne, Noireterre, Audrey, Schaeffer, Laure, Kuhn, Lauriane, Imler, Jean-Luc, Eriani, Gilbert, Meignin, Carine, Martin, Franck, Architecture et Réactivité de l'ARN (ARN), Institut de biologie moléculaire et cellulaire (IBMC), Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS), Modèles Insectes de l'Immunité Innée (M3I), Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut de biologie moléculaire et cellulaire (IBMC), Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS), Plateforme Protéomique Strasbourg - Esplanade (IBMC / CNRS FRC1589 / UNIV Strasbourg), Centre National de la Recherche Scientifique (CNRS)-Institut de Biologie Moléculaire et Cellulaire [Strasbourg] (IBMC), Centre National de la Recherche Scientifique (CNRS), Université de Strasbourg, Investissement d’Avenir program [NetRNA ANR-10-LABX-36, ANR ANR-11-SVSE802501]. Funding for open access charge: Centre National de la Recherche Scientifique., MATHELIN, Sandra, and Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)

Subjects
Base Sequence, Cell-Free System, [SDV]Life Sciences [q-bio], fungi, [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, Internal Ribosome Entry Sites, [SDV.BBM.BM] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, Cell Line, [SDV] Life Sciences [q-bio], Gryllidae, Open Reading Frames, Viral Proteins, Drosophila melanogaster, Protein Biosynthesis, Host-Pathogen Interactions, [SDV.BBM] Life Sciences [q-bio]/Biochemistry, Molecular Biology, Dicistroviridae, Animals, Nucleic Acid Conformation, RNA, Viral, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, 5' Untranslated Regions, Molecular Biology, Ribosomes
Abstract

International audience; Cricket paralysis virus (CrPV) is a dicistrovirus. Its positive-sense single-stranded RNA genome contains two internal ribosomal entry sites (IRESs). The 5' untranslated region (5'UTR) IRES5'UTR mediates translation of non-structural proteins encoded by ORF1 whereas the well-known intergenic region (IGR) IRESIGR is required for translation of structural proteins from open reading frame 2 in the late phase of infection. Concerted action of both IRES is essential for host translation shut-off and viral translation. IRESIGR has been extensively studied, in contrast the IRES5'UTR remains largely unexplored. Here, we define the minimal IRES element required for efficient translation initiation in drosophila S2 cell-free extracts. We show that IRES5'UTR promotes direct recruitment of the ribosome on the cognate viral AUG start codon without any scanning step, using a Hepatitis-C virus-related translation initiation mechanism. Mass spectrometry analysis revealed that IRES5'UTR recruits eukaryotic initiation factor 3, confirming that it belongs to type III class of IRES elements. Using Selective 2'-hydroxyl acylation analyzed by primer extension and DMS probing, we established a secondary structure model of 5'UTR and of the minimal IRES5'UTR. The IRES5'UTR contains a pseudoknot structure that is essential for proper folding and ribosome recruitment. Overall, our results pave the way for studies addressing the synergy and interplay between the two IRES from CrPV.

Published
2017
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3. Zinc Fingers in HIV-1 Gag Precursor Are Not Equivalent for gRNA Recruitment at the Plasma Membrane

Author

Pascal Didier, Raphael Cathagne, Hugues de Rocquigny, Maaz Bin Nasim, Jeremy Bonzi, Roland Marquet, Yves Mély, Philippe Carl, Denis Dujardin, Serena Bernacchi, Halina Anton, Eléonore Réal, Jean-Christophe Paillart, Emmanuel Boutant, Laboratoire de Bioimagerie et Pathologies (LBP), Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS), Architecture et Réactivité de l'ARN (ARN), Institut de biologie moléculaire et cellulaire (IBMC), Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS), Morphogénèse et antigénicité du VIH et du virus des Hépatites (MAVIVH - U1259 Inserm - CHRU Tours ), Centre Hospitalier Régional Universitaire de Tours (CHRU Tours)-Université de Tours (UT)-Institut National de la Santé et de la Recherche Médicale (INSERM), Centre Hospitalier Régional Universitaire de Tours (CHRU TOURS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Tours, and MATHELIN, Sandra

Subjects
[SDV]Life Sciences [q-bio], viruses, Mutant, Biophysics, [SDV.BBM.BM] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, 03 medical and health sciences, 0302 clinical medicine, [SDV.BBM] Life Sciences [q-bio]/Biochemistry, Molecular Biology, Humans, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Guide RNA, 030304 developmental biology, Ribonucleoprotein, Zinc finger, 0303 health sciences, Chemistry, Virus Assembly, Cell Membrane, [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, Zinc Fingers, Genomics, Articles, 3. Good health, Cell biology, [SDV] Life Sciences [q-bio], Cytosol, Membrane, Cytoplasm, HIV-1, RNA, Viral, 030217 neurology & neurosurgery, Intracellular, RNA, Guide, Kinetoplastida
Abstract

International audience; The human immunodeficiency virus type 1 Gag precursor specifically selects the unspliced viral genomic RNA (gRNA) from the bulk of cellular and spliced viral RNAs via its nucleocapsid (NC) domain and drives gRNA encapsidation at the plasma membrane (PM). To further identify the determinants governing the intracellular trafficking of Gag-gRNA complexes and their accumulation at the PM, we compared, in living and fixed cells, the interactions between gRNA and wild-type Gag or Gag mutants carrying deletions in NC zinc fingers (ZFs) or a nonmyristoylated version of Gag. Our data showed that the deletion of both ZFs simultaneously or the complete NC domain completely abolished intracytoplasmic Gag-gRNA interactions. Deletion of either ZF delayed the delivery of gRNA to the PM but did not prevent Gag-gRNA interactions in the cytoplasm, indicating that the two ZFs display redundant roles in this respect. However, ZF2 played a more prominent role than ZF1 in the accumulation of the ribonucleoprotein complexes at the PM. Finally, the myristate group, which is mandatory for anchoring the complexes at the PM, was found to be dispensable for the association of Gag with the gRNA in the cytosol.

Published
2020
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4. Neutrophil Metabolic Shift during Their Lifecycle: Impact on Their Survival and Activation

Author

Injarabian, Louise, Devin, Anne, Ransac, Stéphane, Marteyn, Benoit, MATHELIN, Sandra, Institut de biologie moléculaire et cellulaire (IBMC), Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS), Institut de biochimie et génétique cellulaires (IBGC), Université Bordeaux Segalen - Bordeaux 2-Centre National de la Recherche Scientifique (CNRS), Architecture et Réactivité de l'ARN (ARN), and Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS)

Subjects
energetic metabolism, Cell Survival, [SDV]Life Sciences [q-bio], oxygen sensing, Review, [SDV.BC.BC]Life Sciences [q-bio]/Cellular Biology/Subcellular Processes [q-bio.SC], [SDV.IMM.II]Life Sciences [q-bio]/Immunology/Innate immunity, Neutrophil Activation, infection, Mitochondria, lcsh:Chemistry, lcsh:Biology (General), lcsh:QD1-999, neutrophils, inflammation, [SDV.BC.BC] Life Sciences [q-bio]/Cellular Biology/Subcellular Processes [q-bio.SC], Animals, Humans, nutrient availability, lcsh:QH301-705.5, Oxidation-Reduction, [SDV.IMM.II] Life Sciences [q-bio]/Immunology/Innate immunity, Metabolic Networks and Pathways
Abstract

International audience; Polymorphonuclear neutrophils (PMNs) are innate immune cells, which represent 50% to 70% of the total circulating leukocytes. How PMNs adapt to various microenvironments encountered during their life cycle, from the bone marrow, to the blood plasma fraction, and to inflamed or infected tissues remains largely unexplored. Metabolic shifts have been reported in other immune cells such as macrophages or lymphocytes, in response to local changes in their microenvironment, and in association with a modulation of their pro-inflammatory or anti-inflammatory functions. The potential contribution of metabolic shifts in the modulation of neutrophil activation or survival is anticipated even though it is not yet fully described. If neutrophils are considered to be mainly glycolytic, the relative importance of alternative metabolic pathways, such as the pentose phosphate pathway, glutaminolysis, or the mitochondrial oxidative metabolism, has not been fully considered during activation. This statement may be explained by the lack of knowledge regarding the local availability of key metabolites such as glucose, glutamine, and substrates, such as oxygen from the bone marrow to inflamed tissues. As highlighted in this review, the link between specific metabolic pathways and neutrophil activation has been outlined in many reports. However, the impact of neutrophil activation on metabolic shifts' induction has not yet been explored. Beyond its importance in neutrophil survival capacity in response to available metabolites, metabolic shifts may also contribute to neutrophil population heterogeneity reported in cancer (tumor-associated neutrophil) or auto-immune diseases (Low/High Density Neutrophils). This represents an active field of research. In conclusion, the characterization of neutrophil metabolic shifts is an emerging field that may provide important knowledge on neutrophil physiology and activation modulation. The related question of microenvironmental changes occurring during inflammation, to which neutrophils will respond to, will have to be addressed to fully appreciate the importance of neutrophil metabolic shifts in inflammatory diseases.

Published
2020
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5. RsaI, un ARN régulateur aux multiples facettes, module le métabolisme du pathogène opportuniste Staphylococcus aureus

Author

Desgranges, Emma, Bronesky, Delphine, Corvaglia, Anna, Francois, Patrice, Caballero, Carlos, Prado, Laura, Toledo-Arana, Alejandro, Lasa, Inigo, Moreau, Karen, Vandenesch, François, Marzi, Stefano, Romby, Pascale, Caldelari, Isabelle, Architecture et Réactivité de l'ARN (ARN), Institut de biologie moléculaire et cellulaire (IBMC), Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS), Hôpitaux Universitaires de Genève (HUG), Instituto de Agrobiotecnologıa, Universidad de Navarra [Pamplona] (UNAV), Universidad Pública de Navarra [Espagne] = Public University of Navarra (UPNA), Pathogénie des Staphylocoques – Staphylococcal Pathogenesis (StaPath), Centre International de Recherche en Infectiologie - UMR (CIRI), École normale supérieure - Lyon (ENS Lyon)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-École normale supérieure - Lyon (ENS Lyon)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Architecture et réactivité de l'ARN (ARN), Université Louis Pasteur - Strasbourg I-Centre National de la Recherche Scientifique (CNRS), and MATHELIN, Sandra

Subjects
[SDV] Life Sciences [q-bio], [SDV]Life Sciences [q-bio], [SDV.BBM] Life Sciences [q-bio]/Biochemistry, Molecular Biology, [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, [SDV.BBM.BM] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, ComputingMilieux_MISCELLANEOUS
Abstract

International audience

Published
2019
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6. Thermodynamics of Molecular Machines Using Incremental ITC

Author

Benoit Meyer, Eric Ennifar, Philippe Dumas, Cyrielle Da Veiga, Architecture et Réactivité de l'ARN (ARN), Institut de biologie moléculaire et cellulaire (IBMC), Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS), and MATHELIN, Sandra

Subjects
0301 basic medicine, Computer science, [SDV]Life Sciences [q-bio], Complex formation, [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, Computational biology, 010402 general chemistry, [SDV.BBM.BM] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, 01 natural sciences, Molecular machine, 0104 chemical sciences, [SDV] Life Sciences [q-bio], 03 medical and health sciences, 030104 developmental biology, Atomic resolution, [SDV.BBM] Life Sciences [q-bio]/Biochemistry, Molecular Biology, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, ComputingMilieux_MISCELLANEOUS
Abstract

Molecular biomachines, such as DNA and RNA polymerases or the ribosome, are fascinating biological assemblies able to swiftly perform repeated and highly regulated tasks, with a remarkable accuracy. Significant advances in structural studies during the past 20 years provided a wealth of information regarding their architecture and considerably contributed to a better understanding of their mechanism of action. However, the three-dimensional structure of a biological nanomachine alone does not provide access to its detailed mechanism of action, even when obtained at atomic resolution. When combined with other biophysical approaches, thermodynamic data, together with kinetic data, are essential for a complete description of any binding interaction, revealing forces driving complex formation and providing insights into mechanisms of action. We have developed an incremental ITC approach that is well-suitable for analysis of biomolecular machines. This strategy allows a dissection of molecular biomachine reactions through successive additions in the ITC cell of consecutive substrates.

Published
2019

7. Quantitative and predictive model of kinetic regulation byE. coliTPP riboswitches

Author

Eric Ennifar, Guillaume Hoffmann, Barbara Puffer-Enders, Sondes Guedich, Fabrice Jossinet, Dominique Burnouf, Guillaume Bec, Philippe Dumas, Stéphane Thore, Mireille Baltzinger, Cyrielle Da Veiga, Architecture et Réactivité de l'ARN (ARN), Institut de biologie moléculaire et cellulaire (IBMC), Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS), European Molecular Biology Laboratory Grenoble (EMBL - Outstation ), Acides Nucléiques : Régulations Naturelle et Artificielle (ARNA), Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut de Chimie du CNRS (INC)-Université de Bordeaux (UB), This work was supported in part by the ‘Agence Nationale de la Recherche’ (ANR PCV08_324694)., MATHELIN, Sandra, and Université de Bordeaux (UB)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)

Subjects
pyrophosphate, riboswitches, 0301 basic medicine, Riboswitch, Conformational change, Kinetic Regulation, Operon, [SDV]Life Sciences [q-bio], Aptamer, Biology, [SDV.BBM.BM] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, thiamine, 03 medical and health sciences, chemistry.chemical_compound, Eukaryotic translation, Transcription (biology), RNA polymerase, Escherichia coli, [SDV.BBM] Life Sciences [q-bio]/Biochemistry, Molecular Biology, kinITC, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Molecular Biology, Genetics, Hydroxyl Radical, Alternative splicing, [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, Cell Biology, [SDV] Life Sciences [q-bio], Kinetics, 030104 developmental biology, chemistry, Biophysics, Thermodynamics, Thiamine Pyrophosphate, Research Paper
Abstract

Riboswitches are non-coding elements upstream or downstream of mRNAs that, upon binding of a specific ligand, regulate transcription and/or translation initiation in bacteria, or alternative splicing in plants and fungi. We have studied thiamine pyrophosphate (TPP) riboswitches regulating translation of thiM operon and transcription and translation of thiC operon in E. coli, and that of THIC in the plant A. thaliana. For all, we ascertained an induced-fit mechanism involving initial binding of the TPP followed by a conformational change leading to a higher-affinity complex. The experimental values obtained for all kinetic and thermodynamic parameters of TPP binding imply that the regulation by A. thaliana riboswitch is governed by mass-action law, whereas it is of kinetic nature for the two bacterial riboswitches. Kinetic regulation requires that the RNA polymerase pauses after synthesis of each riboswitch aptamer to leave time for TPP binding, but only when its concentration is sufficient. A quantitative model of regulation highlighted how the pausing time has to be linked to the kinetic rates of initial TPP binding to obtain an ON/OFF switch in the correct concentration range of TPP. We verified the existence of these pauses and the model prediction on their duration. Our analysis also led to quantitative estimates of the respective efficiency of kinetic and thermodynamic regulations, which shows that kinetically regulated riboswitches react more sharply to concentration variation of their ligand than thermodynamically regulated riboswitches. This rationalizes the interest of kinetic regulation and confirms empirical observations that were obtained by numerical simulations.

Published
2016
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8. RsaC sRNA modulates the oxidative stress response of Staphylococcus aureus during manganese starvation

Author

Lalaouna, David, Baude, Jessica, Wu, Zongfu, Tomasini, Arnaud, Chicher, Johana, Marzi, Stefano, Vandenesch, François, Romby, Pascale, Caldelari, Isabelle, Moreau, Karen, Architecture et Réactivité de l'ARN (ARN), Institut de biologie moléculaire et cellulaire (IBMC), Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS), Pathogénie des Staphylocoques – Staphylococcal Pathogenesis (StaPath), Centre International de Recherche en Infectiologie - UMR (CIRI), École normale supérieure - Lyon (ENS Lyon)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-École normale supérieure - Lyon (ENS Lyon)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Nanjing Agricultural University, Plateforme Protéomique Strasbourg Esplanade, Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS), Centre National de Reference des Staphylocoques, Université de Lyon, MATHELIN, Sandra, Nanjing Agricultural University (NAU), Centre International de Recherche en Infectiologie (CIRI), École normale supérieure de Lyon (ENS de Lyon)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Université Jean Monnet - Saint-Étienne (UJM)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-École normale supérieure de Lyon (ENS de Lyon)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Université Jean Monnet - Saint-Étienne (UJM)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), ANR-16-CE11-0007,RIBOSTAPH,La traduction et son contrôle chez Staphylococcus aureus: conséquences sur la virulence et la réponse aux stress(2016), and ANR-17-EURE-0023,IMCBio,Integrative Molecular and Cellular Biology(2017)

Subjects
Manganese, Staphylococcus aureus, Superoxide Dismutase, Gene Expression Regulation, Bacterial, Staphylococcal Infections, Sciences du Vivant [q-bio]/Biochimie, Biologie Moléculaire, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, Sciences du Vivant [q-bio]/Génétique, Oxidative Stress, Bacterial Proteins, Starvation, [SDV.BBM.GTP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Genomics [q-bio.GN], RNA and RNA-protein complexes, [SDV.BBM.GTP] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Genomics [q-bio.GN], Homeostasis, Humans, [SDV.MP.BAC] Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, Oxidation-Reduction
Abstract

International audience; The human opportunistic pathogen Staphylococcus aureus produces numerous small regulatory RNAs (sRNAs) for which functions are still poorly understood. Here, we focused on an atypical and large sRNA called RsaC. Its length varies between different isolates due to the presence of repeated sequences at the 5′ end while its 3′ part is structurally independent and highly conserved. Using MS2-affinity purification coupled with RNA sequencing (MAPS) and quantitative differential proteomics, sodA mRNA was identified as a primary target of RsaC sRNA. SodA is a Mn-dependent superoxide dismutase involved in oxidative stress response. Remarkably, rsaC gene is co-transcribed with the major manganese ABC transporter MntABC and, consequently, RsaC is mainly produced in response to Mn starvation. This 3′UTR-derived sRNA is released from mntABC-RsaC precursor after cleavage by RNase III. The mature and stable form of RsaC inhibits the synthesis of the Mn-containing enzyme SodA synthesis and favors the oxidative stress response mediated by SodM, an alternative SOD enzyme using either Mn or Fe as co-factor. In addition, other putative targets of RsaC are involved in oxidative stress (ROS and NOS) and metal homeostasis (Fe and Zn). Consequently, RsaC may balance two interconnected defensive responses, i.e. oxidative stress and metal-dependent nutritional immunity.

Published
2019
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9. Functional microRNA screen uncovers O-linked N-acetylglucosamine transferase as a host factor modulating hepatitis C virus morphogenesis and infectivity

Author

Hervé Lerat, Jean-Christophe Meunier, Sarah C. Durand, Thomas Baumert, Mirjam B. Zeisel, Laurent Brino, Amélie Weiss, Frank Jühling, Béatrice Chane-Woon-Ming, Catherine Fauvelle, Sophie Pernot, Marion Mercey, Anne Bull, Sébastien Pfeffer, Katharina Herzog, Wolfgang Raffelsberger, Simonetta Bandiera, Institut de Recherche sur les Maladies Virales et Hépatiques (IVH), Université de Strasbourg (UNISTRA)-Institut National de la Santé et de la Recherche Médicale (INSERM), Génétique et épigénétique des maladies métaboliques, neurosensorielles et du développement (Inserm U781), Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM), Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA), Morphogénèse et antigénicité du VIH et du virus des Hépatites (MAVIVH - U1259 Inserm - CHRU Tours ), Centre Hospitalier Régional Universitaire de Tours (CHRU Tours)-Université de Tours (UT)-Institut National de la Santé et de la Recherche Médicale (INSERM), Institut de Génomique d'Evry (IG), Université Paris-Saclay-Institut de Biologie François JACOB (JACOB), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Architecture et Réactivité de l'ARN (ARN), Institut de biologie moléculaire et cellulaire (IBMC), Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS), Institute for Advanced Biosciences / Institut pour l'Avancée des Biosciences (Grenoble) (IAB), Centre Hospitalier Universitaire [Grenoble] (CHU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Etablissement français du sang - Auvergne-Rhône-Alpes (EFS)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), Institut Mondor de Recherche Biomédicale (IMRB), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris-Est Créteil Val-de-Marne - Paris 12 (UPEC UP12)-IFR10, Interactions Virus-Hôte et Maladies Hépatiques, Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut de Virologie, Interaction virus-hôte et maladies du foie, MATHELIN, Sandra, Université de Strasbourg (UNISTRA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Institut National de la Santé et de la Recherche Médicale (INSERM)-IFR10-Université Paris-Est Créteil Val-de-Marne - Paris 12 (UPEC UP12), Université de Tours-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre Hospitalier Régional Universitaire de Tours (CHRU TOURS), Institut de Biologie François JACOB (JACOB), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay, Maladies Virales et Hépatiques, inserm U1110, University of Leipzig (Bioinformatics Group), Universität Leipzig [Leipzig], Virus, pseudo-virus: Morphogénèse et Antigénicité, Université de Tours-EA3856, Institut de génétique et biologie moléculaire et cellulaire (IGBMC), Université Louis Pasteur - Strasbourg I-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Institut für Physik and CINSaT, Universität Kassel, and Universität Kassel [Kassel]

Subjects
[SDV]Life Sciences [q-bio], Cell, molecular mechanisms, Hepacivirus, medicine.disease_cause, Liver disease, 0302 clinical medicine, [SDV.MHEP.MI]Life Sciences [q-bio]/Human health and pathology/Infectious diseases, Gene expression, Sciences du Vivant [q-bio]/Biologie cellulaire, Morphogenesis, ComputingMilieux_MISCELLANEOUS, Host factor, Infectivity, [SDV.MP.VIR] Life Sciences [q-bio]/Microbiology and Parasitology/Virology, 0303 health sciences, Virulence, Gastroenterology, virus diseases, 3. Good health, Cell biology, Up-Regulation, medicine.anatomical_structure, Gene Knockdown Techniques, Host-Pathogen Interactions, Hcv, [SDV.MP.VIR]Life Sciences [q-bio]/Microbiology and Parasitology/Virology, [SDV.MHEP.MI] Life Sciences [q-bio]/Human health and pathology/Infectious diseases, [SDV.BBM.GTP] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Genomics [q-bio.GN], 030211 gastroenterology & hepatology, Hepatitis C virus, Biology, Sciences du Vivant [q-bio]/Médecine humaine et pathologie, N-Acetylglucosaminyltransferases, Sciences du Vivant [q-bio]/Biochimie, Biologie Moléculaire, Article, 03 medical and health sciences, [SDV.BBM.GTP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Genomics [q-bio.GN], microRNA, medicine, hepatocyte, Humans, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, 030304 developmental biology, Life Cycle Stages, [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, [SDV.MHEP.HEG]Life Sciences [q-bio]/Human health and pathology/Hépatology and Gastroenterology, Hepatitis C, Chronic, medicine.disease, [SDV.MHEP.HEG] Life Sciences [q-bio]/Human health and pathology/Hépatology and Gastroenterology, digestive system diseases, MicroRNAs, Gene Expression Regulation, Hepatocytes, hepatitis C, Genome-Wide Association Study
Abstract

ObjectiveInfection of human hepatocytes by the hepatitis C virus (HCV) is a multistep process involving both viral and host factors. microRNAs (miRNAs) are small non-coding RNAs that post-transcriptionally regulate gene expression. Given that miRNAs were indicated to regulate between 30% and 75% of all human genes, we aimed to investigate the functional and regulatory role of miRNAs for the HCV life cycle.DesignTo systematically reveal human miRNAs affecting the HCV life cycle, we performed a two-step functional high-throughput miRNA mimic screen in Huh7.5.1 cells infected with recombinant cell culture-derived HCV. miRNA targeting was then assessed using a combination of computational and functional approaches.ResultsWe uncovered miR-501-3p and miR-619-3p as novel modulators of HCV assembly/release. We discovered that these miRNAs regulate O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT) protein expression and identified OGT and O-GlcNAcylation as regulators of HCV morphogenesis and infectivity. Furthermore, increased OGT expression in patient-derived liver tissue was associated with HCV-induced liver disease and cancer.ConclusionmiR-501-3p and miR-619-3p and their target OGT are previously undiscovered regulatory host factors for HCV assembly and infectivity. In addition to its effect on HCV morphogenesis, OGT may play a role in HCV-induced liver disease and hepatocarcinogenesis.

Published
2019
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10. ITC Studies of Ribosome/Antibiotics Interactions

Author

Eric Ennifar, Emma Schenckbecher, Benoit Meyer, MATHELIN, Sandra, Architecture et Réactivité de l'ARN (ARN), Institut de biologie moléculaire et cellulaire (IBMC), and Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS)

Subjects
0303 health sciences, 030306 microbiology, medicine.drug_class, Bacterial ribosome, [SDV]Life Sciences [q-bio], Antibiotics, Antimicrobial peptides, Translation (biology), [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, Computational biology, Multiresistant bacteria, Biology, [SDV.BBM.BM] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, Ribosome, World health, 3. Good health, [SDV] Life Sciences [q-bio], 03 medical and health sciences, medicine, [SDV.BBM] Life Sciences [q-bio]/Biochemistry, Molecular Biology, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, ComputingMilieux_MISCELLANEOUS, 030304 developmental biology
Abstract

The fight against multiresistant bacteria responsible for nosocomial diseases has recently been classified as an absolute priority by the World Health Organization. For some organisms, priority status has even been assessed as critical, as almost all currently available antibiotics are now inefficient against these "super-bacteria." Ribosome is a major target of several antibiotics, and extensive biochemical and structural studies led to a better understanding of the mechanism of action of drugs targeting translation (Blair et al., Nat Rev Microbiol 13:42-51, 2015; Lin et al., Annu Rev Biochem 87:451-478, 2018; Wilson, Nat Rev Microbiol 12:35-48, 2014; Yonath, Annu Rev Biochem 74:649-79, 2005). However, our knowledge regarding thermodynamic data of compounds targeting the ribosome, which are yet essential for a complete understanding of translation inhibition mechanisms by drugs, is still very poor.In this chapter we describe the use of ITC microcalorimetry to investigate the binding of bacterial ribosome to two antibiotics targeting the peptide tunnel: macrolides and proline-rich antimicrobial peptides (PrAMPs). This strategy yields reliable and artifact-free binding parameters for antibiotics and provides an original view on ribosome/antibiotics interactions.

Published
2019

11. Mapping of Shigella flexneri’s tissue distribution and type III secretion apparatus activity during infection of the large intestine of guinea pigs

Author

F-X Campbell-Valois, Martin Sachse, Giulia Nigro, Philippe J. Sansonetti, Benoit S. Marteyn, Maryse Moya-Nilges, Ellen T. Arena, Pathogénie microbienne moléculaire, Institut Pasteur [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM), University of Wisconsin-Madison, Plateforme BioImagerie Ultrastructurale – Ultrastructural BioImaging Platform (UTechS UBI), Institut Pasteur [Paris], Architecture et Réactivité de l'ARN (ARN), Institut de biologie moléculaire et cellulaire (IBMC), Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS), Chaire Microbiologie et Maladies infectieuses, Collège de France (CdF (institution)), This work was supported by the European Research Council and Howard Hugues Medical Institute (PS) and Canadian Institute of Health Research Project Grant 159517 (FXCV)., MATHELIN, Sandra, Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM), Institut Pasteur [Paris] (IP), Collège de France - Chaire Microbiologie et Maladies infectieuses, and ANR-17-CE15-0012,NEUTROXIA,Effet de l'exposition des neutrophiles à l'oxygène sur leur activation et leur mort : une lame à double tranchant(2017)

Subjects
Microbiology (medical), Colon, Guinea Pigs, Vacuole, medicine.disease_cause, fluorescence microscopy, Microbiology, Type three secretion system, Shigella flexneri, 03 medical and health sciences, 0302 clinical medicine, Genes, Reporter, [SDV.MHEP.MI]Life Sciences [q-bio]/Human health and pathology/Infectious diseases, medicine, Type III Secretion Systems, Immunology and Allergy, Animals, Secretion, Shigella, Tissue Distribution, Intestinal Mucosa, genetically encoded reporters, 030304 developmental biology, Dysentery, Bacillary, 0303 health sciences, Lamina propria, General Immunology and Microbiology, biology, in vivo infection, General Medicine, biology.organism_classification, Mucus, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, 3. Good health, type III secretion system, Disease Models, Animal, Infectious Diseases, medicine.anatomical_structure, Organ Specificity, large intestine, [SDV.MHEP.MI] Life Sciences [q-bio]/Human health and pathology/Infectious diseases, Female, [SDV.MP.BAC] Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, 030217 neurology & neurosurgery, Bacteria, Biomarkers, Research Article
Abstract

Shigella spp. are bacterial pathogens that invade the human colonic mucosa using a type III secretion apparatus (T3SA), a proteinaceous device activated upon contact with host cells. Active T3SAs translocate proteins that carve the intracellular niche of Shigella spp. Nevertheless, the activation state of the T3SA has not been addressed in vivo. Here, we used a green fluorescent protein transcription-based secretion activity reporter (TSAR) to provide a spatio-temporal description of S. flexneri T3SAs activity in the colon of Guinea pigs. First, we observed that early mucus release is triggered in the vicinity of luminal bacteria with inactive T3SA. Subsequent mucosal invasion showed bacteria with active T3SA associated with the brush border, eventually penetrating into epithelial cells. From 2 to 8 h post-challenge, the infection foci expanded, and these intracellular bacteria displayed homogeneously high-secreting activity, while extracellular foci within the lamina propria featured bacteria with low secretion activity. We also found evidence that within lamina propria macrophages, bacteria reside in vacuoles instead of accessing the cytosol. Finally, bacteria were cleared from tissues between 8 and 24 h post-challenge, highlighting the hit-and-run colonization strategy of Shigella. This study demonstrates how genetically encoded reporters can contribute to deciphering pathogenesis in vivo., We mapped Shigella flexneri cells and assessed the activity of their type III secretion apparatus in the large intestine of Guinea pigs.

Published
2019
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12. MS2-Affinity Purification Coupled With RNA Sequencing Approach in the Human Pathogen Staphylococcus aureus

Author

Isabelle Caldelari, David Lalaouna, Emma Desgranges, Stefano Marzi, Architecture et Réactivité de l'ARN (ARN), Institut de biologie moléculaire et cellulaire (IBMC), Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS), Architecture et réactivité de l'ARN (ARN), Université Louis Pasteur - Strasbourg I-Centre National de la Recherche Scientifique (CNRS), and MATHELIN, Sandra

Subjects
0301 basic medicine, Staphylococcus aureus, Virulence Factors, [SDV]Life Sciences [q-bio], Virulence, Human pathogen, Computational biology, Biology, medicine.disease_cause, Interactome, 03 medical and health sciences, medicine, [SDV.BBM] Life Sciences [q-bio]/Biochemistry, Molecular Biology, Humans, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, ComputingMilieux_MISCELLANEOUS, Sequence Analysis, RNA, RNA, Toxic shock syndrome, Gene Expression Regulation, Bacterial, medicine.disease, biology.organism_classification, 3. Good health, [SDV] Life Sciences [q-bio], RNA, Bacterial, 030104 developmental biology, Transfer RNA, Bacteria
Abstract

Staphylococcus aureus is a Gram-positive major human pathogen involved in a wide range of human infectious diseases (from minor skin infections to septicemia, endocarditis or toxic shock syndrome). The treatment of S. aureus infections is very challenging due to the emergence of multiple antibiotic-resistant isolates. The high diversity of clinical symptoms caused by S. aureus depends on the precise expression of numerous virulence factors and stress response pathways, which are tightly regulated at every level (transcriptional, posttranscriptional, translational, and posttranslational). During the last two decades, it has become evident that small regulatory RNAs (sRNAs) play a major role in fast adaptive responses, mainly by targeting mRNA translation. sRNAs act as antisense RNAs by forming noncontiguous pairings with their target mRNAs and their mechanisms of action vary according to the interaction site. To obtain a global and detailed view of the regulatory networks involved in the adaptive processes of S. aureus, we have adapted the MAPS approach to get individual sRNA targetomes. We also set up different strategies to validate MAPS results and establish sRNA regulatory activities. As this method has been first developed in Gram-negative bacteria, we provide here a protocol for its application in S. aureus and highlight underlying differences. Finally, we discuss several points that have been and could be further improved and provide a workflow file for the automatic analysis of the sequencing in Galaxy.

Published
2018

13. Specific Chemical Approaches for Studying Mammalian Ribosomes Complexed with Ligands Involved in Selenoprotein Synthesis

Author

Kossinova, Olga, Malygin, Alexey, Krol, Alain, Karpova, Galina, Architecture et réactivité de l'ARN (ARN), Université Louis Pasteur - Strasbourg I-Centre National de la Recherche Scientifique (CNRS), Institute of of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences, Institute of Chemical Biology and Fundamental Medicine, and MATHELIN, Sandra

Subjects
[SDV] Life Sciences [q-bio], [SDV]Life Sciences [q-bio], [SDV.BBM] Life Sciences [q-bio]/Biochemistry, Molecular Biology, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, ComputingMilieux_MISCELLANEOUS
Abstract

International audience

Published
2018

14. Correction: Structural Insights into Viral Determinants of Nematode Mediated Grapevine fanleaf virus Transmission

Author

Pascale Schellenberger, Claude Sauter, Bernard Lorber, Patrick Bron, Stefano Trapani, Marc Bergdoll, Aurélie Marmonier, Corinne Schmitt-Keichinger, Olivier Lemaire, Gérard Demangeat, Christophe Ritzenthaler, Architecture et réactivité de l'ARN (ARN), Université Louis Pasteur - Strasbourg I-Centre National de la Recherche Scientifique (CNRS), Architecture et Réactivité de l'ARN (ARN), Institut de biologie moléculaire et cellulaire (IBMC), Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS), Centre de Biochimie Structurale [Montpellier] (CBS), Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM)-Institut National de la Santé et de la Recherche Médicale (INSERM), Institut de biologie moléculaire des plantes (IBMP), Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA), Santé de la vigne et qualité du vin (SVQV), Institut National de la Recherche Agronomique (INRA)-Université de Strasbourg (UNISTRA), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS), Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS), MATHELIN, Sandra, Institut National de la Recherche Agronomique (INRA)-Université Louis Pasteur - Strasbourg I, Institut de Recherche Mathématique de Rennes (IRMAR), Université de Rennes 1 (UR1), Université de Rennes (UNIV-RENNES)-Université de Rennes (UNIV-RENNES)-AGROCAMPUS OUEST-Institut National des Sciences Appliquées - Rennes (INSA Rennes), Institut National des Sciences Appliquées (INSA)-Université de Rennes (UNIV-RENNES)-Institut National des Sciences Appliquées (INSA)-École normale supérieure - Rennes (ENS Rennes)-Université de Rennes 2 (UR2), Université de Rennes (UNIV-RENNES)-Centre National de la Recherche Scientifique (CNRS), AGROCAMPUS OUEST, Institut national d'enseignement supérieur pour l'agriculture, l'alimentation et l'environnement (Institut Agro)-Institut national d'enseignement supérieur pour l'agriculture, l'alimentation et l'environnement (Institut Agro)-Université de Rennes 1 (UR1), Université de Rennes (UNIV-RENNES)-Université de Rennes (UNIV-RENNES)-Université de Rennes 2 (UR2), Université de Rennes (UNIV-RENNES)-École normale supérieure - Rennes (ENS Rennes)-Centre National de la Recherche Scientifique (CNRS)-Institut National des Sciences Appliquées - Rennes (INSA Rennes), and Institut National des Sciences Appliquées (INSA)-Université de Rennes (UNIV-RENNES)-Institut National des Sciences Appliquées (INSA)

Subjects
lcsh:Immunologic diseases. Allergy, Nematoda, [SDV]Life Sciences [q-bio], Nepovirus, Static Electricity, Immunology, [SDV.BBM.BM] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, Microbiology, Plant Viruses, 03 medical and health sciences, Capsid, X-Ray Diffraction, Sequence Analysis, Protein, Virology, Genetics, [SDV.BBM] Life Sciences [q-bio]/Biochemistry, Molecular Biology, Animals, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Protein Structure, Quaternary, lcsh:QH301-705.5, Molecular Biology, ComputingMilieux_MISCELLANEOUS, Plant Diseases, 030304 developmental biology, 0303 health sciences, 030302 biochemistry & molecular biology, Correction, [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, [SDV] Life Sciences [q-bio], lcsh:Biology (General), Amino Acid Substitution, Mutation, RNA, Viral, Capsid Proteins, Parasitology, lcsh:RC581-607, Sequence Alignment
Abstract

Many animal and plant viruses rely on vectors for their transmission from host to host. Grapevine fanleaf virus (GFLV), a picorna-like virus from plants, is transmitted specifically by the ectoparasitic nematode Xiphinema index. The icosahedral capsid of GFLV, which consists of 60 identical coat protein subunits (CP), carries the determinants of this specificity. Here, we provide novel insight into GFLV transmission by nematodes through a comparative structural and functional analysis of two GFLV variants. We isolated a mutant GFLV strain (GFLV-TD) poorly transmissible by nematodes, and showed that the transmission defect is due to a glycine to aspartate mutation at position 297 (Gly297Asp) in the CP. We next determined the crystal structures of the wild-type GFLV strain F13 at 3.0 Å and of GFLV-TD at 2.7 Å resolution. The Gly297Asp mutation mapped to an exposed loop at the outer surface of the capsid and did not affect the conformation of the assembled capsid, nor of individual CP molecules. The loop is part of a positively charged pocket that includes a previously identified determinant of transmission. We propose that this pocket is a ligand-binding site with essential function in GFLV transmission by X. index. Our data suggest that perturbation of the electrostatic landscape of this pocket affects the interaction of the virion with specific receptors of the nematode's feeding apparatus, and thereby severely diminishes its transmission efficiency. These data provide a first structural insight into the interactions between a plant virus and a nematode vector.

Published
2017
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15. Genome engineering in the yeast pathogen Candida glabrata using the CRISPR-Cas9 system

Author

Ludovic Enkler, Delphine Richer, Dominique Ferrandon, Fabrice Jossinet, Anthony L. Marchand, Architecture et Réactivité de l'ARN (ARN), Institut de biologie moléculaire et cellulaire (IBMC), Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS), and MATHELIN, Sandra

Subjects
0301 basic medicine, Aspartic Acid Proteases, [SDV]Life Sciences [q-bio], 030106 microbiology, Mutant, Virulence, Candida glabrata, Biology, Protein Serine-Threonine Kinases, [SDV.BBM.BM] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, Genome, Article, Microbiology, Fungal Proteins, 03 medical and health sciences, INDEL Mutation, [SDV.BBM] Life Sciences [q-bio]/Biochemistry, Molecular Biology, CRISPR, Animals, Drosophila Proteins, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Receptors, Immunologic, Homologous Recombination, Gene, Pathogen, Adaptor Proteins, Signal Transducing, Genetics, Fungal protein, Multidisciplinary, [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, biology.organism_classification, Antigens, Differentiation, [SDV] Life Sciences [q-bio], 030104 developmental biology, Drosophila melanogaster, Female, CRISPR-Cas Systems, Genome, Fungal, Genetic Engineering
Abstract

Among Candida species, the opportunistic fungal pathogen Candida glabrata has become the second most common causative agent of candidiasis in the world and a major public health concern. Yet, few molecular tools and resources are available to explore the biology of C. glabrata and to better understand its virulence during infection. In this study, we describe a robust experimental strategy to generate loss-of-function mutants in C. glabrata. The procedure is based on the development of three main tools: (i) a recombinant strain of C. glabrata constitutively expressing the CRISPR-Cas9 system, (ii) an online program facilitating the selection of the most efficient guide RNAs for a given C. glabrata gene, and (iii) the identification of mutant strains by the Surveyor technique and sequencing. As a proof-of-concept, we have tested the virulence of some mutants in vivo in a Drosophila melanogaster infection model. Our results suggest that yps11 and a previously uncharacterized serine/threonine kinase are involved, directly or indirectly, in the ability of the pathogenic yeast to infect this model host organism.

Published
2016
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16. Translational regulation of APOBEC3G mRNA by Vif requires its 5′UTR and contributes to restoring HIV-1 infectivity

Author

Santiago, Guerrero, Camille, Libre, Julien, Batisse, Gaëlle, Mercenne, Delphine, Richer, Géraldine, Laumond, Thomas, Decoville, Christiane, Moog, Roland, Marquet, Jean-Christophe, Paillart, Architecture et Réactivité de l'ARN (ARN), Institut de biologie moléculaire et cellulaire (IBMC), Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS), Aménagement, Développement, Environnement, Santé et Sociétés (ADES), Centre National de la Recherche Scientifique (CNRS)-Université Bordeaux Segalen - Bordeaux 2-Université Bordeaux Montaigne, Architecture et réactivité de l'ARN (ARN), Centre National de la Recherche Scientifique (CNRS)-Université Louis Pasteur - Strasbourg I, Laboratoire de Virologie [Strasbourg], Immuno-Rhumatologie Moléculaire, Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Strasbourg (UNISTRA), Réplication virale, pathogenèse et immunité, Université Louis Pasteur - Strasbourg I-Institut National de la Santé et de la Recherche Médicale (INSERM), Université Bordeaux Segalen - Bordeaux 2-Université Bordeaux Montaigne-Centre National de la Recherche Scientifique (CNRS), Université Louis Pasteur - Strasbourg I-Centre National de la Recherche Scientifique (CNRS), Université de Strasbourg, CNRS, Architecture et Réactivité de l'ARN, UPR9002, F-67000, Strasbourg, France, MATHELIN, Sandra, and Université de Strasbourg (UNISTRA)-Institut National de la Santé et de la Recherche Médicale (INSERM)

Subjects
Gene Expression Regulation, Viral, Proteasome Endopeptidase Complex, viruses, [SDV]Life Sciences [q-bio], virus diseases, HIV Infections, [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, APOBEC-3G Deaminase, biochemical phenomena, metabolism, and nutrition, [SDV.BBM.BM] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, Antiviral Agents, Article, Cytosine Deaminase, [SDV] Life Sciences [q-bio], HEK293 Cells, Mutation, vif Gene Products, Human Immunodeficiency Virus, [SDV.BBM] Life Sciences [q-bio]/Biochemistry, Molecular Biology, Humans, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, RNA, Messenger, 5' Untranslated Regions, Protein Processing, Post-Translational, Alleles, ComputingMilieux_MISCELLANEOUS
Abstract

The essential HIV-1 viral infectivity factor (Vif) allows productive infection of non-permissive cells expressing cytidine deaminases APOBEC3G (A3G) and A3F by decreasing their cellular level, and preventing their incorporation into virions. Unlike the Vif-induced degradation of A3G, the functional role of the inhibition of A3G translation by Vif remained unclear. Here, we show that two stem-loop structures within the 5′-untranslated region of A3G mRNA are crucial for translation inhibition by Vif in cells, and most Vif alleles neutralize A3G translation efficiently. Interestingly, K26R mutation in Vif abolishes degradation of A3G by the proteasome but has no effect at the translational level, indicating these two pathways are independent. These two mechanisms, proteasomal degradation and translational inhibition, similarly contribute to decrease the cellular level of A3G by Vif and to prevent its incorporation into virions. Importantly, inhibition of A3G translation is sufficient to partially restore viral infectivity in the absence of proteosomal degradation. These findings demonstrate that HIV-1 has evolved redundant mechanisms to specifically inhibit the potent antiviral activity of A3G.

Published
2016
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17. Ribosomal 18S rRNA base pairs with mRNA during eukaryotic translation initiation

Author

Angelita Simonetti, Franck Martin, Quentin Vicens, Jean-François Ménétret, S. Kundhavai Natchiar, Lydia Prongidi-Fix, Bruno P. Klaholz, Gilbert Eriani, Alexander G. Myasnikov, Architecture et Réactivité de l'ARN (ARN), Institut de biologie moléculaire et cellulaire (IBMC), Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS), Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA), This work was supported by ANR-2011-svse8-02501 (MITIC project), the European Research Council (ERC Starting Grant N_243296 TRANSLATIONMACHINERY) and the Centre National pour la Recherche Scientifique (CNRS). The electron microscope facility was supported by the Alsace Region, the FRM, INSERM, CNRS and the Association pour la Recherche sur le Cancer (ARC) and by the French Infrastructure for Integrated Structural Biology (FRISBI) ANR-10-INSB-05-01, and Instruct as part of the European Strategy Forum on Research Infrastructures (ESFRI)., ANR-11-BSV8-0025,MITIC,Messagers dont l'Initiation de la Traduction Interne est dirigée par la Coiffe(2011), ANR-10-INBS-0005,FRISBI,Infrastructure Française pour la Biologie Structurale Intégrée(2010), European Project: 243296,EC:FP7:ERC,ERC-2009-StG,TRANSLATIONMACHINERY(2009), Université de Strasbourg (UNISTRA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), MATHELIN, Sandra, BLANC - Messagers dont l'Initiation de la Traduction Interne est dirigée par la Coiffe - - MITIC2011 - ANR-11-BSV8-0025 - BLANC - VALID, Infrastructure Française pour la Biologie Structurale Intégrée - - FRISBI2010 - ANR-10-INBS-0005 - INBS - VALID, and Integrative structure and function study of the bacterial and human protein synthesis machinery. - TRANSLATIONMACHINERY - - EC:FP7:ERC2009-12-01 - 2014-11-30 - 243296 - VALID

Subjects
Models, Molecular, 0301 basic medicine, Science, Kozak consensus sequence, [SDV]Life Sciences [q-bio], Codon, Initiator, General Physics and Astronomy, Biology, [SDV.BBM.BM] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, Ribosome, Article, General Biochemistry, Genetics and Molecular Biology, Histones, Mice, 03 medical and health sciences, Eukaryotic translation, Start codon, Eukaryotic initiation factor, RNA, Ribosomal, 18S, [SDV.BBM] Life Sciences [q-bio]/Biochemistry, Molecular Biology, Animals, Eukaryotic Small Ribosomal Subunit, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, RNA, Messenger, Peptide Chain Initiation, Translational, Base Pairing, Genetics, Multidisciplinary, Base Sequence, [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, General Chemistry, Ribosomal RNA, Stem-loop, [SDV] Life Sciences [q-bio], 030104 developmental biology, Mutation, Nucleic Acid Conformation, Rabbits, Ribosomes
Abstract

Eukaryotic mRNAs often contain a Kozak sequence that helps tether the ribosome to the AUG start codon. The mRNA of histone H4 (h4) does not undergo classical ribosome scanning but has evolved a specific tethering mechanism. The cryo-EM structure of the rabbit ribosome complex with mouse h4 shows that the mRNA forms a folded, repressive structure at the mRNA entry site on the 40S subunit next to the tip of helix 16 of 18S ribosomal RNA (rRNA). Toe-printing and mutational assays reveal that an interaction exists between a purine-rich sequence in h4 mRNA and a complementary UUUC sequence of helix h16. Together the present data establish that the h4 mRNA harbours a sequence complementary to an 18S rRNA sequence which tethers the mRNA to the ribosome to promote proper start codon positioning, complementing the interactions of the 40S subunit with the Kozak sequence that flanks the AUG start codon., Prokaryotic translation initiation involves mRNA-ribosomal RNA base pairing interactions. Here, the authors provide evidence for a similar base pairing interactions occurring between the human h4 mRNA and helix 16 of the small subunit rRNA to position the correct AUG codon in the decoding site.

Published
2016
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18. Identification of factors involved in target RNA-directed microRNA degradation

Author

Haas, Gabrielle, Cetin, Semih, Messmer, Mélanie, Chane-Woon-Ming, Béatrice, Terenzi, Olivier, Chicher, Johana, Kuhn, Lauriane, Hammann, Philippe, Pfeffer, Sébastien, Architecture et Réactivité de l'ARN (ARN), Institut de biologie moléculaire et cellulaire (IBMC), Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS), Institut de Génomique d'Evry (IG), Université Paris-Saclay-Institut de Biologie François JACOB (JACOB), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Plateforme Protéomique Strasbourg Esplanade, Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS), Laboratoire d'étude de la dynamique des protéomes (LEDyP), Université Joseph Fourier - Grenoble 1 (UJF)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM), Plate-forme de protéomique [Strasbourg Grand Est], Institut de Biologie Moléculaire et Cellulaire (IBMC), Etude de la dynamique des protéomes (EDyP ), Laboratoire de Biologie à Grande Échelle (BGE - UMR S1038), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), and MATHELIN, Sandra

Subjects
0301 basic medicine, Small RNA, RNA Stability, [SDV]Life Sciences [q-bio], Molecular Sequence Data, Trans-acting siRNA, Cytomegalovirus, Computational biology, Biology, [SDV.BBM.BM] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, Mice, 03 medical and health sciences, Cell Line, Tumor, microRNA, [SDV.BBM] Life Sciences [q-bio]/Biochemistry, Molecular Biology, Genetics, Animals, Humans, Gene silencing, Biotinylation, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, RNA, Messenger, ComputingMilieux_MISCELLANEOUS, mRNA Cleavage and Polyadenylation Factors, Base Sequence, Polynucleotide Adenylyltransferase, RNA, [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, Oligonucleotides, Antisense, Argonaute, Nucleotidyltransferases, [SDV] Life Sciences [q-bio], MicroRNAs, RNA silencing, HEK293 Cells, 030104 developmental biology, Gene Expression Regulation, Argonaute Proteins, Cytomegalovirus Infections, Exoribonucleases, Hepatocytes, HeLa Cells
Abstract

The mechanism by which micro (mi)RNAs control their target gene expression is now well understood. It is however less clear how the level of miRNAs themselves is regulated. Under specific conditions, abundant and highly complementary target RNA can trigger miRNA degradation by a mechanism involving nucleotide addition and exonucleolytic degradation. One such mechanism has been previously observed to occur naturally during viral infection. To date, the molecular details of this phenomenon are not known. We report here that both the degree of complementarity and the ratio of miRNA/target abundance are crucial for the efficient decay of the small RNA. Using a proteomic approach based on the transfection of biotinylated antimiRNA oligonucleotides, we set to identify the factors involved in target-mediated miRNA degradation. Among the retrieved proteins, we identified members of the RNA-induced silencing complex, but also RNA modifying and degradation enzymes. We further validate and characterize the importance of one of these, the Perlman Syndrome 3'-5' exonuclease DIS3L2. We show that this protein interacts with Argonaute 2 and functionally validate its role in target-directed miRNA degradation both by artificial targets and in the context of mouse cytomegalovirus infection.

Published
2016
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19. eIF3 Peripheral Subunits Rearrangement after mRNA Binding and Start-Codon Recognition

Author

Adeline Renaud, A. Myasnikov, Lauriane Kuhn, Angelita Simonetti, Yaser Hashem, Jailson Brito Querido, Eder Mancera-Martínez, MATHELIN, Sandra, Initiative d'excellence - Par-delà les frontières, l'Université de Strasbourg - - UNISTRA2010 - ANR-10-IDEX-0002 - IDEX - VALID, Architecture et Réactivité de l'ARN (ARN), Institut de biologie moléculaire et cellulaire (IBMC), Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS), Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Université de Strasbourg (UNISTRA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Plateforme Protéomique Strasbourg - Esplanade (IBMC / CNRS FRC1589 / UNIV Strasbourg), Centre National de la Recherche Scientifique (CNRS)-Institut de Biologie Moléculaire et Cellulaire [Strasbourg] (IBMC), This work was supported by the ANR grant @RAction program ‘‘ANR CryoEM80S’’ and the LABEX: ANR-10-LABX-0036_NETRNA and benefits from funding from the state managed by the French National Research Agency as part of the Investments for the Future program (to Y.H.)., ANR-10-IDEX-0002,UNISTRA,Par-delà les frontières, l'Université de Strasbourg(2010), and Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA)

Subjects
Models, Molecular, 0301 basic medicine, MESH: Eukaryotic Initiation Factor-3, Five prime untranslated region, MESH: Eukaryotic Initiation Factor-2, Protein Conformation, Eukaryotic Initiation Factor-3, MESH: Codon, Initiator, [SDV]Life Sciences [q-bio], Eukaryotic Initiation Factor-2, Eukaryotic Initiation Factor-1, Codon, Initiator, MESH: Rabbits, beta-Globins, [SDV.BBM.BM] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, MESH: Structure-Activity Relationship, MESH: Protein Conformation, RNA, Transfer, Eukaryotic initiation factor, MESH: Animals, MESH: Protein Subunits, [SDV] Life Sciences [q-bio], MESH: Nucleic Acid Conformation, ribosome, MESH: Protein Biosynthesis, Transfer RNA, Rabbits, eIF3, MESH: Eukaryotic Initiation Factor-1, MESH: Models, Molecular, Protein Binding, eukaryotic translation initiation, Biology, Structure-Activity Relationship, 03 medical and health sciences, Eukaryotic translation, [SDV.BBM] Life Sciences [q-bio]/Biochemistry, Molecular Biology, Animals, Humans, MESH: Protein Binding, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, RNA, Messenger, Molecular Biology, eIF3 peripheral subunits, MESH: RNA, Messenger, Binding Sites, MESH: Humans, MESH: beta-Globins, [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, Cell Biology, MESH: Multiprotein Complexes, MESH: RNA, Transfer, Molecular biology, Ribosomal binding site, Protein Subunits, Internal ribosome entry site, 030104 developmental biology, MESH: Binding Sites, Multiprotein Complexes, Protein Biosynthesis, eIF4A, Nucleic Acid Conformation, cryo-EM, Ribosomes, MESH: Ribosomes
Abstract

International audience; mRNA translation initiation in eukaryotes requires the cooperation of a dozen eukaryotic initiation factors (eIFs) forming several complexes, which leads to mRNA attachment to the small ribosomal 40S subunit, mRNA scanning for start codon, and accommodation of initiator tRNA at the 40S P site. eIF3, composed of 13 subunits, 8 core (a, c, e, f, h, l, k, and m) and 5 peripheral (b, d, g, i, and j), plays a central role during this process. Here we report a cryo-electron microscopy structure of a mammalian 48S initiation complex at 5.8 Å resolution. It shows the relocation of subunits eIF3i and eIF3g to the 40S intersubunit face on the GTPase binding site, at a late stage in initiation. On the basis of a previous study, we demonstrate the relocation of eIF3b to the 40S intersubunit face, binding below the eIF2-Met-tRNAi(Met) ternary complex upon mRNA attachment. Our analysis reveals the deep rearrangement of eIF3 and unravels the molecular mechanism underlying eIF3 function in mRNA scanning and timing of ribosomal subunit joining.

Published
2016
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20. Experimental Approaches to Study Genome Packaging of Influenza A Viruses

Author

Catherine Isel, Nadia Naffakh, Sandie Munier, Architecture et réactivité de l'ARN (ARN), Université Louis Pasteur - Strasbourg I-Centre National de la Recherche Scientifique (CNRS), Génétique Moléculaire des Virus à ARN - Molecular Genetics of RNA Viruses (GMV-ARN (UMR_3569 / U-Pasteur_2)), Institut Pasteur [Paris]-Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS), This study was supported by PREDEMICS (Grant Agreement No. 278433)., European Project: 278433,EC:FP7:HEALTH,FP7-HEALTH-2011-two-stage,PREDEMICS(2011), Institut Pasteur [Paris] (IP)-Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS), MATHELIN, Sandra, and Preparedness, Prediction and Prevention of Emerging Zoonotic Viruses with Pandemic Potential using Multidisciplinary Approaches - PREDEMICS - - EC:FP7:HEALTH2011-11-01 - 2016-10-31 - 278433 - VALID

Subjects
0301 basic medicine, Electron Microscope Tomography, Viral budding, viruses, lcsh:QR1-502, packaging assay, Review, Biology, medicine.disease_cause, Genome, lcsh:Microbiology, influenza virus, 03 medical and health sciences, Microscopy, Electron, Transmission, Virology, Influenza A virus, medicine, Humans, In Situ Hybridization, Fluorescence, Ribonucleoprotein, [SDV.MP.VIR] Life Sciences [q-bio]/Microbiology and Parasitology/Virology, Budding, single-molecule FISH, Virus Assembly, packaging signal, RNA-RNA interaction, Reverse genetics, Reverse Genetics, Single Molecule Imaging, 3. Good health, Cell biology, competitive reverse genetics, 030104 developmental biology, Infectious Diseases, Cytoplasm, [SDV.MP.VIR]Life Sciences [q-bio]/Microbiology and Parasitology/Virology, Function (biology)
Abstract

International audience; The genome of influenza A viruses (IAV) consists of eight single-stranded negative sense viral RNAs (vRNAs) encapsidated into viral ribonucleoproteins (vRNPs). It is now well established that genome packaging (i.e., the incorporation of a set of eight distinct vRNPs into budding viral particles), follows a specific pathway guided by segment-specific cis-acting packaging signals on each vRNA. However, the precise nature and function of the packaging signals, and the mechanisms underlying the assembly of vRNPs into sub-bundles in the cytoplasm and their selective packaging at the viral budding site, remain largely unknown. Here, we review the diverse and complementary methods currently being used to elucidate these aspects of the viral cycle. They range from conventional and competitive reverse genetics, single molecule imaging of vRNPs by fluorescence in situ hybridization (FISH) and high-resolution electron microscopy and tomography of budding viral particles, to solely in vitro approaches to investigate vRNA-vRNA interactions at the molecular level.

Published
2016
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21. Structure of the 70S ribosome from human pathogenStaphylococcus aureus

Author

Pascale Romby, Anthony Bochler, Quentin Vicens, François Grosse, I. Khusainov, Yaser Hashem, Stefano Marzi, Gulnara Yusupova, Jean-François Ménétret, A. Myasnikov, Johana Chicher, Marat Yusupov, Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Université de Strasbourg (UNISTRA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Architecture et réactivité de l'ARN (ARN), Université Louis Pasteur - Strasbourg I-Centre National de la Recherche Scientifique (CNRS), Institut de génétique et biologie moléculaire et cellulaire (IGBMC), Université Louis Pasteur - Strasbourg I-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Plateforme Protéomique Strasbourg Esplanade, Institut de biologie moléculaire et cellulaire (IBMC), Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS), MATHELIN, Sandra, Architecture et Réactivité de l'ARN (ARN), and Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS)

Subjects
Protein Conformation, alpha-Helical, 0301 basic medicine, [SDV]Life Sciences [q-bio], Gene Expression, Human pathogen, [SDV.BBM.BM] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, medicine.disease_cause, Ribosome, 0302 clinical medicine, Structural Biology, Translational regulation, Pathogen, ComputingMilieux_MISCELLANEOUS, 0303 health sciences, Translation (biology), [SDV] Life Sciences [q-bio], RNA, Bacterial, Staphylococcus aureus, Corrigendum, Bacillus subtilis, Protein Binding, Ribosomal Proteins, Computational biology, Molecular Dynamics Simulation, Biology, Microbiology, 03 medical and health sciences, Antibiotic resistance, Bacterial Proteins, Species Specificity, Escherichia coli, [SDV.BBM] Life Sciences [q-bio]/Biochemistry, Molecular Biology, Genetics, medicine, Protein Interaction Domains and Motifs, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Amino Acid Sequence, 030304 developmental biology, Binding Sites, Sequence Homology, Amino Acid, Thermus thermophilus, Cryoelectron Microscopy, RNA, [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, 030104 developmental biology, Protein Biosynthesis, Nucleic Acid Conformation, Protein Conformation, beta-Strand, Ribosomes, Sequence Alignment, 030217 neurology & neurosurgery
Abstract

Comparative structural studies of ribosomes from various organisms keep offering exciting insights on how species-specific or environment-related structural features of ribosomes may impact translation specificity and its regulation. Although the importance of such features may be less obvious within more closely related organisms, their existence could account for vital yet species-specific mechanisms of translation regulation that would involve stalling, cell survival and antibiotic resistance. Here, we present the first full 70S ribosome structure from Staphylococcus aureus, a Gram-positive pathogenic bacterium, solved by cryo-electron microscopy. Comparative analysis with other known bacterial ribosomes pinpoints several unique features specific to S. aureus around a conserved core, at both the protein and the RNA levels. Our work provides the structural basis for the many studies aiming at understanding translation regulation in S. aureus and for designing drugs against this often multi-resistant pathogen.

Published
2016
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22. The ribosome prohibits the G•U wobble geometry at the first position of the codon–anticodon helix

Author

Gulnara Yusupova, A. Rozov, Eric Westhof, Marat Yusupov, Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Université de Strasbourg (UNISTRA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Architecture et Réactivité de l'ARN (ARN), Institut de biologie moléculaire et cellulaire (IBMC), Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS), French National Research Agency [ANR-15-CE11-0021-01 to G.Y., E.W], European Research Council advanced grant [294312 to M.Y.]. Funding for open access charge: French National Research Agency [ANR-15-CE11-0021-01]., ANR-15-CE11-0021,RFS,Fidélité et structure du ribosome étudiée par analyse aux rayons X.(2015), European Project: 294312,EC:FP7:ERC,ERC-2011-ADG_20110310,SBPSSHS(2012), MATHELIN, Sandra, Fidélité et structure du ribosome étudiée par analyse aux rayons X. - - RFS2015 - ANR-15-CE11-0021 - AAPG2015 - VALID, and Structural Basis of Protein Synthesis System of Human Cell - SBPSSHS - - EC:FP7:ERC2012-04-01 - 2017-03-31 - 294312 - VALID

Subjects
Models, Molecular, 0301 basic medicine, Speed wobble, Base pair, [SDV]Life Sciences [q-bio], Geometry, Biology, Crystallography, X-Ray, [SDV.BBM.BM] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, Ribosome, 03 medical and health sciences, RNA, Transfer, Structural Biology, RNA, Ribosomal, 16S, Anticodon, [SDV.BBM] Life Sciences [q-bio]/Biochemistry, Molecular Biology, Genetics, Protein biosynthesis, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, RNA, Messenger, Codon, Guanosine, 030102 biochemistry & molecular biology, Thermus thermophilus, [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, Translation (biology), Ribosomal RNA, [SDV] Life Sciences [q-bio], 030104 developmental biology, Protein Biosynthesis, Transfer RNA, Helix, Nucleic Acid Conformation, Ribosomes
Abstract

International audience; Precise conversion of genetic information into proteins is essential to cellular health. However, a margin of error exists and is at its highest on the stage of translation of mRNA by the ribosome. Here we present three crystal structures of 70S ribosome complexes with messenger RNA and transfer RNAs and show that when a G•U base pair is at the first position of the codon-anticodon helix a conventional wobble pair cannot form because of inescapable steric clash between the guanosine of the A codon and the key nucleotide of decoding center adenosine 1493 of 16S rRNA. In our structure the rigid ribosomal decoding center, which is identically shaped for cognate or near-cognate tRNAs, forces this pair to adopt a geometry close to that of a canonical G•C pair. We further strengthen our hypothesis that spatial mimicry due either to base tautomerism or ionization dominates the translation infidelity mechanism.

Published
2016
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